ABSTRACT
OBJECTIVE: To assess the effect of caspase 3 inhibition, in the expression of intracellular antigens induced by apoptosis. MATERIAL AND METHODS: Skin explants of neonatal Balb/c mice were used to assess the autoantigen expression. Skin was obtained by punch biopsies, tissues were cultured in DMEM; cell death was induced by chemicals and assessed by TUNEL. The expression of La, Ro, Sm, RNP, Cajal Bodies and NuMa antigens were monitored by immunohistochemistry using autoantibodies or monoclonal antibodies against these antigens. RESULTS: Chemicals used to induce cell death, successfully produced apoptosis or necrosis in more than 60% of keratinocytes, and viability was significantly decreased when it was compared with those in controls. An increased expression of all skin intracellular antigens in skin biopsies treated with chemicals, major antigenic expression was detected with anti-La and anti-Ro antibodies. The caspase 3 inhibitor DEVD-CMK significantly decreased the expression of antigens induced by chemicals. CONCLUSION: By this result we can infer that caspase inhibitors modify apoptosis and decrease the autoantigens associated to cell death.
Subject(s)
Amino Acid Chloromethyl Ketones/pharmacology , Apoptosis/immunology , Autoantigens/biosynthesis , Autoimmune Diseases/prevention & control , Caspase Inhibitors , Cysteine Proteinase Inhibitors/therapeutic use , Skin/immunology , Animals , Animals, Newborn , Autoimmune Diseases/etiology , Biopsy , Camptothecin/pharmacology , Cells, Cultured/drug effects , Cells, Cultured/enzymology , Cells, Cultured/immunology , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Drug Evaluation, Preclinical , Hydrogen Peroxide/pharmacology , In Situ Nick-End Labeling , Mercuric Chloride/pharmacology , Mice , Mice, Inbred BALB C , Organ Culture Techniques , Skin/enzymologyABSTRACT
OBJECTIVE: Examine the presence of functional inducible nitric oxide synthase (iNOS) in lupus nephritis lesions. METHODS: Seventeen kidney biopsies from patients with lupus nephritis and an equal number of normal control kidney biopsies were examined for the presence of iNOS and endothelial nitric oxide synthase (eNOS) and citrulline by using immunohistochemical methods. Additionally, iNOS and eNOS mRNAs were examined by reverse transcription -PCR amplification of total renal RNA. RESULTS: All biopsies expressed constitutive eNOS, but in contrast to normal kidney biopsies, 70% of the lupus biopsies also expressed iNOS mRNA and the cognate protein. Eight positive biopsies corresponded to class IV lupus nephritis, which also had a high degree of citrullination. CONCLUSIONS: The data indicate that functional iNOS activity is present in glomeruli as part of the inflammatory process in the kidney; therefore the products of iNOS could play a role in the pathogenesis of lupus nephritis.
Subject(s)
Citrulline/metabolism , Kidney , Lupus Nephritis/metabolism , Lupus Nephritis/pathology , Nitric Oxide Synthase Type II/metabolism , Adolescent , Adult , Animals , Biopsy , Female , Humans , Kidney/metabolism , Kidney/pathology , Lupus Nephritis/classification , Male , Mice , Mice, Inbred BALB C , Nitric Oxide , Nitric Oxide Synthase Type III/metabolism , Young AdultABSTRACT
The present investigation assesses the possible role of apoptosis and necrosis in intracellular antigen exposure of kidneys from Balb/c mice. Renal tissues were cultured and treated with chemicals to induce apoptosis and /or necrosis. The expression of intracellular antigens Sm, RNP, Ro and La were monitored with antibodies against these antigens. Main results confirm that renal intracellular antigens are released and exposed onto the surface of apoptotic and necrotic cells, therefore these antigens become an easy target of autoantibodies. This mechanism may be important in the lupus nephritis pathogenesis.
Subject(s)
Autoantigens/biosynthesis , Kidney/immunology , Kidney/pathology , Ribonucleoproteins, Small Nuclear/metabolism , Ribonucleoproteins/metabolism , Animals , Animals, Newborn , Apoptosis/drug effects , Mice , Mice, Inbred BALB C , Necrosis/chemically induced , Tissue Culture Techniques , snRNP Core Proteins , SS-B AntigenABSTRACT
OBJECTIVE: Present study addresses the issue whether apoptosis and necrosis increases the antigenicity of proteins recognized by antinuclear antibodies. MATERIAL AND METHODS: HEp-2 cells were cultured in standard conditions; apoptosis was induced by camptothecin and necrosis by mercuric chloride. Protein antigenicity of cell extracts was tested onto nitrocellulose membranes and probed with positive or negative sera for antinuclear antibodies by a luminescent-dot-ELISA system. RESULTS: Apoptotic changes in HEp-2 cells appeared by 24 hours of camptothecin exposure, meanwhile the necrotic features become visible earlier. Luminescence was significantly superior in ANA positive sera than in ANA negative controls. Antinuclear antibody sera recognized better the antigens from the apoptotic and necrotic cells than controls without chemical treatments. CONCLUSIONS: Apoptosis and necrosis increase the ANA binding by better availability of intracellular antigens, or by disclosing cryptic epitopes.
